The invention relates to the development of PCR primers and primer mixtures.
In our experience with PCR amplification of cpn60 universal target sequences from genomic DNA templates from individual organisms, we have found that templates containing at least 57% G+C are problematic and are usually difficult or impossible to amplify with conventional primers H279/H280 or H729/H730 (the so-called cpn60“universal primers” or “inosine primers” or “I primers”). Chaperonin-60(cpn60) proteins are sequence-related molecular chaperones found in prokaryotes and in the plastids and mitochondria of eukaryotes.
High G+C content organisms include organisms of significance to agriculture, human and animal health, industry and the environment. They are important members of microbial communities. Given the status quo, the inability to amplify partial cpn60 sequences from these types of organisms makes them inaccessible to present methods, preventing the development of methods to detect and identify them or to take stock of them in complex microbial communities.
More specifically, the problems are that inosine is not a “neutral” base, inosine-based primers bind less efficiently with increasing G+C content and such primers fail to amplify from templates with G+C content of greater than 58% in the cpn60 universal target region.